Dual effector population modification gene-drive strains of the African malaria mosquitoes, Anopheles gambiae and Anopheles coluzzii.

Proposed genetic approaches for reducing human malaria include population modification, which introduces genes into vector mosquitoes to reduce or prevent parasite transmission. We demonstrate the potential of Cas9/guide RNA (gRNA)-based gene-drive systems linked to dual antiparasite effector genes to spread rapidly through mosquito populations. Two strains have an autonomous gene-drive system coupled to dual anti-Plasmodium falciparum effector genes comprising single-chain variable fragment monoclonal antibodies targeting parasite ookinetes and sporozoites in the African malaria mosquitoes Anopheles gambiae (AgTP13) and Anopheles coluzzii (AcTP13). The gene-drive systems achieved full introduction within 3 to 6 mo after release in small cage trials. Life-table analyses revealed no fitness loads affecting AcTP13 gene-drive dynamics but AgTP13 males were less competitive than wild types. The effector molecules reduced significantly both parasite prevalence and infection intensities. These data supported transmission modeling of conceptual field releases in an island setting that shows meaningful epidemiological impacts at different sporozoite threshold levels (2.5 to 10 k) for human infection by reducing malaria incidence in optimal simulations by 50 to 90% within as few as 1 to 2 mo after a series of releases, and by ≥90% within 3 mo. Modeling outcomes for low sporozoite thresholds are sensitive to gene-drive system fitness loads, gametocytemia infection intensities during parasite challenges, and the formation of potentially drive-resistant genome target sites, extending the predicted times to achieve reduced incidence. TP13-based strains could be effective for malaria control strategies following validation of sporozoite transmission threshold numbers and testing field-derived parasite strains. These or similar strains are viable candidates for future field trials in a malaria-endemic region.

Safety, efficacy, and feasibility of percutaneous transhepatic endoscopic holmium laser lithotripsy for bile duct stones.

OBJECTIVE: To evaluate the safety and efficacy of percutaneous transhepatic endoscopic holmium laser lithotripsy (PTEHL) for patients with intrahepatic bile duct (IHBD) and common bile duct (CBD) stones. MATERIAL AND METHODS: This retrospective study included 530 patients (mean age: 55.6 ± 8.5; 64.2% female) with IHBD and/or CBD stones at a single institution from January 2019 to December 2021. PTEHL was the chosen treatment for patients with large, complex stones, or those for whom Endoscopic Retrograde Cholangiopancreatography (ERCP) failed or presented difficulties. Patients showing signs of cholangitis required pre-PTEHL drainage. Stone clearance was confirmed by post-procedural cholangiography, and the technique was deemed successful when target stones were removed. Complications were recorded according to the Society of Interventional Radiology adverse event classification. RESULTS: The mean stone size was 20.9 ± 11.9 mm, multiple stones observed in 460 patients (86.8%). A total of 225 patients (42.5%) had stones in both the IHBD and CBD; biliary-enteric anastomosis in 50 patients (9.4%). ERCP for stone removal proved unsuccessful in 18 patients (3.4%). Pre-IHBD drainage was performed in 271 patients (51.1%). The majority (488 patients, 92.1%) underwent a single PTEHL session. The technique was successful in 523 patients (98.7%), with 7 patients requiring surgery due to unsuccessful target stone removal. Complications were noted in 75 patients (14.2%), including 4.7% with severe complications and 9.4% with minor complications. CONCLUSION: PTEHL is a safe and effective method for the treatment of both intrahepatic and extrahepatic bile duct stones. This approach is a valuable option for complex stone cases, particularly when ERCP is unsuccessful or encounters significant challenges. CLINICAL RELEVANCE STATEMENT: Percutaneous Transhepatic Endoscopic Holmium Laser Lithotripsy is a safe and effective treatment method for intrahepatic and extrahepatic biliary stones, particularly in cases of complex stones. KEY POINTS: Percutaneous transhepatic biliary stone removal is difficult for large or intraductal stones. Percutaneous Transhepatic Endoscopic Holmium Laser Lithotripsy (PTEHL) demonstrated a high success rate with few major complications. PTEHL can treat biliary stones, particularly stones that are difficult or have failed ERCP treatment.

Digital Physical Activity and Exercise Interventions for People Living with Chronic Kidney Disease: A Systematic Review of Health Outcomes and Feasibility.

Physical activity is essential to interrupt the cycle of deconditioning associated with chronic kidney disease (CKD). However, access to targeted physical activity interventions remain under-supported due to limited funding and specialised staff. Digital interventions may address some of these factors. This systematic review sought to examine the evidence base of digital interventions focused on promoting physical activity or exercise and their effect on health outcomes for people living with CKD. Electronic databases (PubMed, CINAHL, Embase, Cochrane) were searched from 1 January 2000 to 1 December 2023. Interventions (smartphone applications, activity trackers, websites) for adults with CKD (any stage, including transplant) which promoted physical activity or exercise were included. Study quality was assessed, and a narrative synthesis was conducted. Of the 4057 records identified, eight studies (five randomised controlled trials, three single-arm studies) were included, comprising 550 participants. Duration ranged from 12-weeks to 1-year. The findings indicated acceptability and feasibility were high, with small cohort numbers and high risk of bias. There were inconsistent measures of physical activity levels, self-efficacy, body composition, physical function, and psychological outcomes which resulted in no apparent effects of digital interventions on these domains. Data were insufficient for meta-analysis. The evidence for digital interventions to promote physical activity and exercise for people living with CKD is limited. Despite popularity, there is little evidence that current digital interventions yield the effects expected from traditional face-to-face interventions. However, 14 registered trials were identified which may strengthen the evidence-base.

Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae.

A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, Anopheles gambiae The drive system targets the cardinal gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro, with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.

A Depth-Based Hybrid Approach for Safe Flight Corridor Generation in Memoryless Planning.

This paper presents a depth-based hybrid method to generate safe flight corridors for a memoryless local navigation planner. It is first proposed to use raw depth images as inputs in the learning-based object-detection engine with no requirement for map fusion. We then employ an object-detection network to directly predict the base of polyhedral safe corridors in a new raw depth image. Furthermore, we apply a verification procedure to eliminate any false predictions so that the resulting collision-free corridors are guaranteed. More importantly, the proposed mechanism helps produce separate safe corridors with minimal overlap that are suitable to be used as space boundaries for path planning. The average intersection of union (IoU) of corridors obtained by the proposed algorithm is less than 2%. To evaluate the effectiveness of our method, we incorporated it into a memoryless planner with a straight-line path-planning algorithm. We then tested the entire system in both synthetic and real-world obstacle-dense environments. The obtained results with very high success rates demonstrate that the proposed approach is highly capable of producing safe corridors for memoryless local planning.

Experimental population modification of the malaria vector mosquito, Anopheles stephensi.

Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (≤ 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.

Low-cost and versatile analytical tool with purpose-made capillary electrophoresis coupled to contactless conductivity detection: Application to antibiotics quality control in Vietnam.

In this study, the development of our purpose-made capacitively coupled contactless conductivity detection (C4 D) for CE is reported. These systems have been employed as a simple, versatile, and cost-effective analytical tool. CE-C4 D devices, whose principle is based on the control of the ion movements under an electrical field, can be constructed even with a modest financial budget and limited infrastructure. A featured application was developed for quality control of antimicrobial drugs using CE-C4 D, with most recent work on determination of aminoglycoside and glycopeptide antibiotics being communicated. For aminoglycosides, the development of CE-C4 D methods was adapted to two categories. The first one includes drugs (liquid or powder form) for intravenous injection, containing either amikacin, streptomycin, kanamycin A, or kanamycin B. The second one covers drugs for eye drops (liquid or ointment form), containing either neomycin, tobramycin, or polymyxin. The CE-C4 D method development was also made for determination of some popular glycopeptide antibiotics in Vietnam, including vancomycin and teicoplanin. The best detection limit achieved using the developed CE-C4 D methods was 0.5 mg/L. Good agreement between results from CE-C4 D and the confirmation method (HPLC- Photometric Diode Array ) was achieved, with their result deviations less than 8% and 13% for aminoglycoside and glycopeptide antibiotics, respectively.

Characterisation of 12 microsatellite loci in the Vietnamese commercial clam Lutraria rhynchaena Jonas 1844 (Heterodonta: Bivalvia: Mactridae) through next-generation sequencing.

The marine clam Lutraria rhynchaena is gaining popularity as an aquaculture species in Asia. Lutraria populations are present in the wild throughout Vietnam and several stocks have been established and translocated for breeding and aquaculture grow-out purposes. In this study, we demonstrate the feasibility of utilising Illumina next-generation sequencing technology to streamline the identification and genotyping of microsatellite loci from this clam species. Based on an initial partial genome scan, 48 microsatellite markers with similar melting temperatures were identified and characterised. The 12 most suitable polymorphic loci were then genotyped using 51 individuals from a population in Quang Ninh Province, North Vietnam. Genetic variation was low (mean number of alleles per locus = 2.6; mean expected heterozygosity = 0.41). Two loci showed significant deviation from Hardy-Weinberg equilibrium (HWE) and the presence of null alleles, but there was no evidence of linkage disequilibrium among loci. Three additional populations were screened (n = 7-36) to test the geographic utility of the 12 loci, which revealed 100 % successful genotyping in two populations from central Vietnam (Nha Trang). However, a second population from north Vietnam (Co To) could not be successfully genotyped and morphological evidence and mitochondrial variation suggests that this population represents a cryptic species of Lutraria. Comparisons of the Qang Ninh and Nha Trang populations, excluding the 2 loci out of HWE, revealed statistically significant allelic variation at 4 loci. We reported the first microsatellite loci set for the marine clam Lutraria rhynchaena and demonstrated its potential in differentiating clam populations. Additionally, a cryptic species population of Lutraria rhynchaena was identified during initial loci development, underscoring the overlooked diversity of marine clam species in Vietnam and the need to genetically characterise population representatives prior to microsatellite development. The rapid identification and validation of microsatellite loci using next-generation sequencing technology warrant its integration into future microsatellite loci development for key aquaculture species in Vietnam and more generally, aquaculture countries in the South East Asia region.

A Pandemic Innovation: Student-Led Model to Increase Accessibility to Educational and Mentorship Opportunities.

The Canadian Ophthalmology Student Interest Group (COSIG) is the first national medical student-led specialty interest group in Canada. COSIG has run several initiatives aimed at increasing students' opportunities for ophthalmology exposure and learning, including a resident-medical student mentorship program, an annual 6-week introductory course, amongst other events, and webinars.

Preferences of people with chronic kidney disease regarding digital health interventions that promote healthy lifestyle: qualitative systematic review with meta-ethnography.

OBJECTIVES: Diet and physical activity are crucial for people with chronic kidney disease (CKD) to maintain good health. Digital health interventions can increase access to lifestyle services. However, consumers' perspectives are unclear, which may reduce the capacity to develop interventions that align with specific needs and preferences. Therefore, this review aims to synthesise the preferences of people with CKD regarding digital health interventions that promote healthy lifestyle. DESIGN: Qualitative systematic review with meta-ethnography. DATA SOURCES: Databases Scopus, CENTRAL, MEDLINE, CINAHL and SPORTDiscus were searched between 2000 and 2023. ELIGIBILITY CRITERIA: Primary research papers that used qualitative exploration methods to explore the preferences of adults with CKD (≥18 years) regarding digital health interventions that promoted diet, physical activity or a combination of these health behaviours. DATA EXTRACTION AND SYNTHESIS: Two independent reviewers screened title, abstract and full text. Discrepancies were resolved by a third reviewer. Consumers' quotes were extracted verbatim and synthesised into higher-order themes and subthemes. RESULTS: Database search yielded 5761 records. One record was identified following communication with a primary author. 15 papers were included. These papers comprised 197 consumers (mean age 51.0±7.2), including 83 people with CKD 1-5; 61 kidney transplant recipients; 53 people on dialysis. Sex was reported in 182 people, including 53% male. Five themes were generated regarding consumers' preferences for digital lifestyle interventions. These included simple instruction and engaging design; individualised interventions; virtual communities of care; education and action plans; and timely reminders and automated behavioural monitoring. CONCLUSION: Digital health interventions were considered an important mechanism to access lifestyle services. Consumers' preferences are important to ensure future interventions are tailored to specific needs and goals. Future research may consider applying the conceptual framework of consumers' preferences in this review to develop and evaluate the effect of a digital lifestyle intervention on health outcomes. PROSPERO REGISTRATION NUMBER: CRD42023411511.

Complementary ensemble clustering of biomedical data.

The rapidly growing availability of electronic biomedical data has increased the need for innovative data mining methods. Clustering in particular has been an active area of research in many different application areas, with existing clustering algorithms mostly focusing on one modality or representation of the data. Complementary ensemble clustering (CEC) is a recently introduced framework in which Kmeans is applied to a weighted, linear combination of the coassociation matrices obtained from separate ensemble clustering of different data modalities. The strength of CEC is its extraction of information from multiple aspects of the data when forming the final clusters. This study assesses the utility of CEC in biomedical data, which often have multiple data modalities, e.g., text and images, by applying CEC to two distinct biomedical datasets (PubMed images and radiology reports) that each have two modalities. Referent to five different clustering approaches based on the Kmeans algorithm, CEC exhibited equal or better performance in the metrics of micro-averaged precision and Normalized Mutual Information across both datasets. The reference methods included clustering of single modalities as well as ensemble clustering of separate and merged data modalities. Our experimental results suggest that CEC is equivalent or more efficient than comparable Kmeans based clustering methods using either single or merged data modalities.

Validation of a telephone-based administration of the simplified nutritional appetite questionnaire.

BACKGROUND: Anorexia of aging is characterized by an age-associated reduction of appetite, whose aetiology in most cases is multifactorial and which often triggers malnutrition. The Simplified Nutritional Appetite Questionnaire (SNAQ) is an established screening tool. This study aimed to investigate reliability, validity, and feasibility of its telephone administration (T-SNAQ) in German community-dwelling older adults. METHODS: This cross-sectional single-centre study recruited participants from April 2021 to September 2021. First, the SNAQ was translated into German according to an established methodology. After translation, reliability, construct validity, and feasibility of the T-SNAQ were analysed. A convenience sample of community-dwelling older adults aged ≥70 years was recruited. The following measurements were applied to all participants: T-SNAQ, Mini Nutritional Assessment - Short Form (MNA-SF), six-item Katz index of independence in activities of daily living (ADL), eight-item Lawton instrumental activities of daily living (IADL), telephone Montreal Cognitive Assessment (T-MoCA); FRAIL scale, Geriatric Depression Scale (GDS-15) and Charlson co-morbidity index as well as daily caloric and protein intake. RESULTS: One hundred twenty participants (59.2% female) with a mean age of 78.0 ± 5.8 years were included in the present study. The percentage of participants identified with poor appetite based on T-SNAQ was 20.8% (n = 25). T-SNAQ showed a good internal reliability with a Cronbach's alpha coefficient of 0.64 and a good test-retest reliability [intraclass coefficient of 0.95 (P < 0.05)]. Regarding construct validity, T-SNAQ was significantly positively correlated with MNA-SF (r = 0.213), T-MoCA (r = 0.225), daily energy (r = 0.222) and protein intake (r = 0.252) (P < 0.05). It also demonstrated a significant negative association with GDS-15 (r = -0.361), FRAIL scale (r = -0.203) and Charlson co-morbidity index (r = -0.272). Regarding applicability, the mean time for T-SNAQ was 95 s and completion rate was 100%. CONCLUSIONS: The T-SNAQ is a feasible screening instrument for anorexia of aging in community-dwelling older adults via telephone interviews.

Percutaneous transhepatic endoscopic thulium laser vaporesection for management of severe and focal benign biliary strictures.

Background: Biliary lithiasis and strictures in the bile ducts have a causality. Dilation or stent placement is routinely used to treat strictures but fibrosis can lead to their recurrence. Thulium laser vaporesection with percutaneous transhepatic endoscopy is a novel therapeutic modal-ity for managing severe, focal benign biliary strictures (BBSs). There are few reports about this method of treating BBSs. Our study aimed to determine the safety and efficacy of this technique. Methods: Fifteen patients (six males and nine females) with BBSs underwent stricture ablation with thulium laser via percutaneous transhepatic endoscopy. The immediate and short-term technical success and complication rates were evaluated. Results: Biliary strictures appeared in segmental branches of two patients, in the left or right hepatic duct of twelve patients, and in the common bile duct of one patient. The immediate and short-term technical success rates of the thulium laser procedure were 100%. The lumen of the strictures measured 1-3 mm before the procedure and improved to 4-5 mm in six (40%) patients, 5-10 mm in five (33.3%) patients, and 10-15 mm in four (26.7%) patients after the procedure. No mortality and major procedure-related complications were observed. One patient experienced a minor complication (hemobilia). Conclusions: Percutaneous transhepatic endoscopic thulium laser ablation appears to be safe and effective for treating short-segment BBSs. However, further studies with large samples and long follow-up periods are necessary to fully determine the long-term outcomes of this technique.

Lifestyle Interventions, Kidney Disease Progression, and Quality of Life: A Systematic Review and Meta-analysis.

Rationale & Objective: Poor dietary patterns and low physical activity levels are important lifestyle-related factors that contribute to negative health outcomes in individuals with chronic kidney disease (CKD). Previous systematic reviews have not explicitly focused on these lifestyle factors, nor undertaken meta-analyses of any effects. We aimed to evaluate the effect of lifestyle interventions (such as diet, exercise, and other lifestyle-related interventions) on the risk factors for and progression of CKD and the quality of life. Study Design: Systematic review and meta-analysis. Setting & Study Populations: Individuals aged 16 years or older with CKD stages 1 to 5 not requiring kidney replacement therapy. Selection Criteria for Studies: Randomized controlled trials of interventions. Data Extraction: Kidney function, albuminuria, creatinine, systolic blood pressure, diastolic blood pressure, body weight, glucose control, and quality of life. Analytical Approach: A random-effects meta-analysis with evidence certainty assessed using GRADE. Results: Seventy-eight records describing 68 studies were included. Twenty-four studies (35%) were dietary interventions, 23 (34%) exercise, 9 (13%) behavioral, 1 (2%) hydration, and 11 (16%) multiple component. Lifestyle interventions resulted in significant improvements in creatinine (weighted mean difference [WMD], -0.43 mg/dL; 95% CI, -0.74 to -0.11; P = 0.008); 24-hour albuminuria (WMD, -53 mg/24 h; 95% CI, -56 to -50; P < 0.001); systolic blood pressure (WMD, -4.5 mm Hg; 95% CI, -6.7 to -2.4; P < 0.001); diastolic blood pressure (WMD, -2.2 mm Hg; 95% CI, -3.7 to -0.8; P = 0.003); and body weight (WMD, -1.1 kg; 95% CI, -2.0 to -0.1; P = 0.025). Lifestyle interventions did not result in significant changes in the estimated glomerular filtration rate (0.9 mL/min/1.73 m2; 95% CI, -0.6 to 2.3; P = 0.251). However, narrative synthesis indicated that lifestyle intervention resulted in improvements in the quality of life. Limitations: Certainty of the evidence was rated very low for most outcomes, primarily owing to the risk of bias and inconsistency. No meta-analysis was possible for quality-of-life outcomes because of variations in measurement tools. Conclusions: Lifestyle interventions seem to positively affect some risk factors for progression of CKD and quality of life.

Data Informing Governing Body Resistance-Training Guidelines Exhibit Sex Bias: An Audit-Based Review.

The objective of this review was to examine the ratio of female and male participants utilised in data informing consensus statements and position stands in the field of resistance training (RT). In order to achieve this objective, we conducted an 'audit' style review. We accessed three databases: SPORTDiscus, MEDLINE and Google Scholar utilising the following search terms: resistance or strength training AND consensus statements or position statements/stands. Eligibility criteria included consensus statements and position stands for RT in youth, adults and older adults. In this paper we have used the term 'female' to describe biological sex. Gender is a social construct and often describes roles and behaviours that society assigns to men or women. In this paper we have used the term 'women' to describe gender. Reference lists from each guideline were screened with the number of male and female participants extracted from each study. We also extracted data on the gender of the authors of the statements. We located 11 guidelines encompassing a total of 104,251,363 participants. Youth guidelines were comprised of 69% male participants. There were 287 studies that included both sexes, 205 male-only and 92 female-only studies. Adult guidelines were comprised of 70% male participants. There were 104 studies that included both sexes, 240 male-only and 44 female-only studies. Older adult guidelines were comprised of 54% female participants. There were 395 studies that included both sexes, 112 male-only and 83 female-only studies. Women authors comprised 13% of all authors of position stands and consensus statements. These results demonstrate an under-representation of females and women as both participants and as authors. It is imperative to ensure that data informing governing body guidelines and consensus statements are representative of the population they aim to inform. If this is not possible, guidelines should clearly state when their data and recommendations are based primarily upon one sex.

Discovery of Ligands for TRIM58, a Novel Tissue-Selective E3 Ligase.

Redirecting E3 ligases to neo-substrates, leading to their proteasomal disassembly, known as targeted protein degradation (TPD), has emerged as a promising alternative to traditional, occupancy-driven pharmacology. Although the field has expanded tremendously over the past years, the choice of E3 ligases remains limited, with an almost exclusive focus on CRBN and VHL. Here, we report the discovery of novel ligands to the PRY-SPRY domain of TRIM58, a RING ligase that is specifically expressed in erythroid precursor cells. A DSF screen, followed by validation using additional biophysical methods, led to the identification of TRIM58 ligand TRIM-473. A basic SAR around the chemotype was established by utilizing a competitive binding assay employing a short FP peptide probe derived from an endogenous TRIM58 substrate. The X-ray co-crystal structure of TRIM58 in complex with TRIM-473 gave insights into the binding mode and potential exit vectors for bifunctional degrader design.

Cas9-mediated maternal effect and derived resistance alleles in a gene-drive strain of the African malaria vector mosquito, Anopheles gambiae.

CRISPR/Cas9 technologies are important tools for the development of gene-drive systems to modify mosquito vector populations to control the transmission of pathogens that cause diseases such as malaria. However, one of the challenges for current Cas9-based drive systems is their ability to produce drive-resistant alleles resulting from insertions and deletions (indels) caused principally by nonhomologous end-joining following chromosome cleavage. Rapid increases in the frequency of such alleles may impair gene-drive dynamics. We explored the generation of indels in the germline and somatic cells in female gene-drive lineages using a series of selective crosses between a gene-drive line, AgNosCd-1, and wild-type mosquitoes. We find that potential drive-resistant mutant alleles are generated largely during embryonic development, most likely caused by deposition of the Cas9 endonuclease and guide RNAs in oocytes and resulting embryos by homozygous and hemizygous gene-drive mothers.

Commercial Potential of the Cyanobacterium Arthrospira maxima: Physiological and Biochemical Traits and the Purification of Phycocyanin.

Arthrospira maxima is a natural source of fine chemicals for multiple biotechnological applications. We determined the optimal environmental conditions for A. maxima by measuring its relative growth rate (RGR), pigment yield, and photosynthetic performance under different pH and temperature conditions. RGR was highest at pH 7-9 and 30 °C. Chlorophyll a, phycocyanin, maximal quantum yield (Fv/Fm), relative maximal electron transport rate (rETRmax), and effective quantum yield (ΦPSII) were highest at pH 7-8 and 25 °C. Interestingly, phycoerythrin and allophycocyanin content was highest at 15 °C, which may be the lowest optimum temperature reported for phycobiliprotein production in the Arthrospira species. A threestep purification of phycocyanin (PC) by ultrafiltration, ion-exchange chromatography, and gel filtration resulted in a 97.6% purity of PC.

Beyond the eye: Kynurenine pathway impairment causes midgut homeostasis dysfunction and survival and reproductive costs in blood-feeding mosquitoes.

Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes.

Microinjection Method for Anopheles gambiae Embryos.

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.